Atrial natriuretic peptide family is composed of three members--ANF, BNP and CNP. These are the most hypotensive agents known to date. They work through their receptors, which are also membrane guanylate cyclases. ANF-RGC is a receptor of ANF and BNP, and CNP-RGC of CNP. The signal transduction machinery of both ANF-RGC and CNPRGC is almost identical. The peptide hormone binds at the extracellular domain of the cyclase and activates its intracellular catalytic domain. ATP is obligatory for the transduction machinery of ANF-RGC and CNP-RGC. The overall objective of the proposed research is to elucidate at the molecular level the mechanism by which ATP modulates the signal transduction activities of ANF-RGC and CNP-RGC. ATP exhibits its transduction activity by directly binding to the cyclase. The domain that mediates ATP-dependent signaling has been identified and termed the ATP regulatory module (ARM). Recently, the three-dimensional structure of the ARM domain, including its ATP binding pocket in ANF-RGC, has been resolved through computer modeling and its functional validity in the ATPdirected transduction step/s has been confirmed through point mutation studies. Besides revealing the three dimensional configuration of the ATP-binding pocket, the ARM domain model shows an intriguing ATP-induced feature. ATP induces the exposure of a hydrophobic region in the ARM domain, which is distant to the ATP-binding pocket and is involved in the activation of the cyclase. Based on these features of the model, the proposed specific aims of the research address two most fundamental issues. The proposed AIM 1 is to validate the model-predicted ATP binding domain by direct ATP binding studies. In these studies the ARM domain will be cross-linked with ATP, proteolytically cleaved, the ATP-bound fragment(s) will be purified and its identity determined by protein sequencing. AIM 2 is designed to validate the importance of the ATP-induced hydrophobic region in activation of the catalytic domain of ANF-RGC. This will be achieved through point mutation/expression studies. The recent gene-knock out studies show that ANF/ANF-RGC signal transduction system is directly involved in overcoming the salt-sensitive hypertension. Therefore, understanding the basic steps of the molecular machinery of ANF-RGC will contribute in developing therapy for hypertension and other heart-related diseases.